The effect of COVID rehabilitation for ongoing symptoms Post HOSPitalisation with COVID-19 (PHOSP-R): protocol for a randomised parallel group controlled trial on behalf of the PHOSP consortium

Introduction Many adults hospitalised with COVID-19 have persistent symptoms such as fatigue, breathlessness and brain fog that limit day-to-day activities. These symptoms can last over 2 years. Whilst there is limited controlled studies on interventions that can support those with ongoing symptoms, there has been some promise in rehabilitation interventions in improving function and symptoms either using face-to-face or digital methods, but evidence remains limited and these studies often lack a control group. Methods and analysis This is a nested single-blind, parallel group, randomised control trial with embedded qualitative evaluation comparing rehabilitation (face-to-face or digital) to usual care and conducted within the PHOSP-COVID study. The aim of this study is to determine the effectiveness of rehabilitation interventions on exercise capacity, quality of life and symptoms such as breathlessness and fatigue. The primary outcome is the Incremental Shuttle Walking Test following the eight week intervention phase. Secondary outcomes include measures of function, strength and subjective assessment of symptoms. Blood inflammatory markers and muscle biopsies are an exploratory outcome. The interventions last eight weeks and combine symptom-titrated exercise therapy, symptom management and education delivered either in a face-to-face setting or through a digital platform (www.yourcovidrecovery.nhs.uk). The proposed sample size is 159 participants, and data will be intention-to-treat analyses comparing rehabilitation (face-to-face or digital) to usual care. Ethics and dissemination Ethical approval was gained as part of the PHOSP-COVID study by Yorkshire and the Humber Leeds West Research NHS Ethics Committee, and the study was prospectively registered on the ISRCTN trial registry (ISRCTN13293865). Results will be disseminated to stakeholders, including patients and members of the public, and published in appropriate journals. Article summary Strengths and limitations of this study • This protocol utilises two interventions to support those with ongoing symptoms of COVID-19 • This is a two-centre parallel-group randomised controlled trial • The protocol has been supported by patient and public involvement groups who identified treatments of symptoms and activity limitation as a top priority Supplementary Information The online version contains supplementary material available at 10.1186/s13063-023-07093-7.

usual gait speed and lower limb strength which are measured by a three stage balance test (feet shoulder width apart, semi-tandem, and tandem position), 4m gait speed test, and a five repetition sit-to-stand test, respectively 1 . Participants can score a maximum of 12 points from the three assessments, with a higher score indicating a greater level of physical function.

Muscle strength assessments
Maximum handgrip strength will be measured using a dynamometer (JAMAR, UK), performed three times on both the dominant and non-dominant hand. The best score is taken per hand. Maximal isometric quadriceps strength will be measured using an isometric dynamometer on the dominant leg. The participant will have three practice attempts at 50%, 60% and 80% effort and three true attempts at 100% effort. The best attempt will be analysed.

Physical activity and sleep
Physical activity will be measured using wrist worn accelerometers worn on the nondominant wrist (GENEActive Original, Activinsights Ltd, Kimbolton, UK) 24 hours per day, for a total of four weeks. This will be broken down into 2 x 14-day periods. The first period will be one week before and one week into the intervention. The second period will be during the final week of the intervention and the week after completing the intervention. A valid physical activity measurement is defined as a minimum of three days, with a valid day defined as at least 16 hours of wear 2 . Measures of physical activity such as average step count, total physical activity, intensity distribution, time inactive, in light-intensity and moderate-to-vigorous intensity, and the intensity of the most active 5, 10 and 30 minutes of the day will be extracted. In addition, total sleep time, the duration of the sleep window and sleep efficiency will be derived. Accelerometer data will be processed using the open-source R-package GGIR 3 . The data will then be cleaned as previously reported 4 .

Questionnaires
The following questionnaires will be used to assess patient self-reported health-status pre-

Assessment of inflammatory markers
This is an optional outcome measure for patients. Before and after the intervention period, venous blood samples will be drawn into blood collection tubes containing EDTA and sodium heparin as anticoagulants. This will be in a sub-group of patients randomised to either the control or face-to-face group, and will be an optional part of the trial. Further details are available in the online supplement. Heparinised whole blood will be used for flow cytometric determination of immune cell subsets and for isolation of peripheral blood mononuclear cells (PBMCs) for stimulation with lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB). EDTA whole blood will be used for analysis of total and differential leukocyte counts with an automated haematology analyser.

Flow cytometry
Monocytes and lymphocytes will be gated based on forward vs. side scatter area and fluorescently conjugated antibodies will be used to identify classical, intermediate and nonclassical monocyte subsets, T cells (including helper and cytotoxic subsets) and their naïve and memory subsets, and pro-and anti-senolytic NK cells (Table 2). Fluorescence minus one controls will be used to gate the abovementioned subsets. The proportions of the different subsets will be used with differential leukocyte counts to calculate the circulating numbers for each subset. Samples will be prepared using standard staining methods and data will be acquired using a 4-colour flow cytometer (Accuri C6, BD, Oxford, UK).

Peripheral Blood Monocyte Cell (PMBC) isolation and stimulation
Heparinised whole blood diluted in a 1:1 ratio with Dulbecco's phosphate buffered saline (D-PBS) will be layered onto Histopaque-1077, from which PBMCs will be derived using density gradient centrifugation. The number of viable PBMCs in the sample will be determined using a standard haemocytometer and trypan blue staining. On a sterile 24-well cell culture plate PBMCs in cell culture media will be seeded at 170,000 cells per well and treated with 100ng/ml of either LPS or SEB. After incubation for 24h at 37.0°C, 5% CO 2 , the supernatants will be collected and frozen at -80°C for later determination of TNF-α, IL-6, and IL-10 concentrations using enzyme-linked immunosorbent assay (ELISA) kits.